(S)-Gossypol acetic acid binds with high affinity to the BH3-binding groove of Bcl-xL and Bcl-2 proteins. These anti-apoptotic proteins are members of the Bcl-2 family that regulate mitochondrial outer membrane permeabilization and apoptosis. By occupying the BH3-binding pocket, (S)-Gossypol displaces pro-apoptotic BH3-only proteins (such as Bid, Bim, and Bad), thereby activating Bax and Bak to initiate mitochondrial outer membrane permeabilization, cytochrome c release, and caspase-dependent apoptosis. The compound shows selectivity for Bcl-2 and Bcl-xL over other Bcl-2 family members such as Mcl-1, making it a valuable tool for studying apoptosis regulation in cancer cells.

There are two enantiomers of natural racemic gossypol: (-)-gossypol and (S)-gossypol ((+)-gossypol). The binding affinities of (+)- and (-)-gossypol to Bcl-2 or Bcl-xL are identical. It is more effective for causing apoptosis and suppressing cell proliferation to use (-)-gossypol rather than (+)-gossypol. In a 6-day MTT experiment, the racemic form of gossypol and each enantiomer were evaluated against UM-SCC-6 and UM-SCC-14A. (-)-gossypol showed a higher degree of growth inhibition in comparison to (±)-gossypol than (+)-gossypol in both examined cell lines (P<0.001). The growth inhibitory impact of (±)-gossypol is moderate, however it is only noticeable at higher dosages (10 μM, P<0.0001) [1].

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(S)-Gossypol acetic acid potently induces cell death in Jurkat cells overexpressing Bcl-2 with an IC₅0 of 18.1 microM, and in cells overexpressing Bcl-xL with an IC₅0 of 22.9 microM. The compound demonstrates concentration-dependent cytotoxicity in various cancer cell lines including leukemia, lymphoma, breast, prostate, and lung cancer cells. It effectively induces apoptosis through the intrinsic mitochondrial pathway, as evidenced by cytochrome c release, caspase-3 activation, and PARP cleavage. The (S)-enantiomer shows superior potency compared to the (R)-enantiomer and racemic gossypol, attributed to its higher binding affinity to the BH3-binding groove of Bcl-2 family proteins.

In vivo, (S)-Gossypol acetic acid has demonstrated antitumor activity in various xenograft mouse models. The compound exhibits oral bioavailability and can be administered orally or intraperitoneally. In murine tumor models, treatment with (S)-Gossypol results in significant tumor growth inhibition, reduced tumor volume, and increased apoptosis in tumor tissues. The compound has been evaluated in combination with other chemotherapeutic agents, showing synergistic or additive antitumor effects. In preclinical models of prostate, breast, and lung cancers, (S)-Gossypol effectively reduces tumor burden and prolongs survival. The compound also shows activity against Bcl-2-overexpressing tumors that are resistant to conventional chemotherapy.

In vitro binding assays for (S)-Gossypol typically involve fluorescence polarization or surface plasmon resonance (SPR) techniques using recombinant Bcl-2 or Bcl-xL proteins. In fluorescence polarization assays, a fluorescently labeled BH3 peptide (e.g., FITC-Bim BH3) is incubated with recombinant Bcl-2 or Bcl-xL protein in assay buffer. Varying concentrations of (S)-Gossypol are added to compete with the labeled peptide for binding to the BH3-binding groove. After incubation at room temperature for 30-60 minutes, fluorescence polarization is measured using a plate reader. IC₅0 values are calculated from competition curves. Alternatively, isothermal titration calorimetry (ITC) can be used to directly measure binding affinity (Kd) and thermodynamic parameters.

Cellular assays for (S)-Gossypol acetic acid are performed using cancer cell lines expressing Bcl-2 or Bcl-xL, such as Jurkat, HL-60, or MCF-7 cells. Cells are cultured in appropriate medium and seeded in 96-well plates. Following overnight attachment, cells are treated with serial dilutions of the compound (typically 0-100 microM) for 24-72 hours. Cell viability is assessed using MTT, XTT, or CellTiter-Glo assays. Apoptosis is quantified using Annexin V-FITC/propidium iodide staining followed by flow cytometry. Caspase-3/7 activity is measured using fluorogenic substrates. Western blotting is performed to assess PARP cleavage, cytochrome c release, and expression of Bcl-2 family proteins. IC₅0 values are calculated from dose-response curves.

In vivo efficacy studies for (S)-Gossypol are conducted using immunocompromised mice bearing subcutaneous xenografts of human cancer cell lines (e.g., HL-60, MCF-7, PC-3). Tumors are established by injecting 5-10 × 10⁶ cells into the flank of nude or SCID mice. When tumors reach approximately 100-200 mm3, mice are randomized into treatment groups (n=6-10 per group). (S)-Gossypol acetic acid is administered orally at doses of 25-100 mg/kg daily or intraperitoneally at 10-50 mg/kg. Tumor volume and body weight are monitored 2-3 times per week. At study termination, tumors are excised, weighed, and processed for histological analysis (H&E, TUNEL, immunohistochemistry for cleaved caspase-3).

Absorption, Distribution and Excretion
Fat-soluble gossypol is readily absorbed from the gastrointestinal tract. It is highly bound to amino acids (especially lysine) and dietary iron. Gossypol's binding, metabolism, and urinary excretion are limited; most is excreted in feces.

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Pharmacokinetic properties of (S)-Gossypol have been studied in preclinical models. The compound exhibits moderate oral bioavailability (approximately 20-40%) with peak plasma concentrations (Cmax) achieved 2-4 hours post-administration. It shows extensive plasma protein binding (>95%) and a long elimination half-life (t1/2 of 10-20 hours in rodents). The compound is metabolized primarily in the liver via CYP450 enzymes and excreted in bile and feces. (S)-Gossypol acetic acid (CAS#: 1189561-66-7) has a molecular weight of 578.61 and formula C32H34O10. It is soluble in DMSO and ethanol, with limited aqueous solubility. The acetic acid salt form improves solubility and handling characteristics for in vivo formulations.

Toxicity Summary
Gossypol may induce apoptosis by modulating Bax and Bcl-2 proteins. It is also an inhibitor of calcineurin and protein kinase C and has been shown to bind to calmodulin. (L1239) Interactions …This study used roosters (n = 144) from different humoral immune selection strains. Three individuals from each strain were randomly selected and placed in cages and fed a corn-soybean meal (control) diet for 14 days. Then, six cages from each strain were randomly selected and given four different dietary treatments (1000 mg/kg gossypol, 1000 mg/kg silymarin, a mixture of 1000 mg/kg gossypol and silymarin, or a control diet). Body weight and feed intake data were collected for 21 consecutive days, and blood was collected weekly to collect plasma and determine hematocrit. The chickens were then euthanized, and livers were collected for histological and enzyme activity analysis. Weekly endpoints were analyzed using repeated measures and regression analysis. Plasma and liver enzyme activities, as well as histological parameters, were analyzed using analysis of variance (ANOVA). No significant interactions were observed between diets and strains. Chickens fed gossypol and gossypol-silymarin diets ceased gaining weight on day 14 (P < 0.001) and experienced weight loss on day 21 (P < 0.001). These chickens also showed elevated γ-glutamyltransferase levels on day 14; by day 21, their activity further increased (P < 0.001). Histological examination of liver sections revealed significant fatty degeneration (P < 0.001). Furthermore, quinone reductase activities were significantly higher in chickens treated with gossypol and the combined gossypol-silymarin diet compared to the control and silymarin-treated groups (P < 0.001). Silymarin did not alleviate any clinical symptoms of gossypol poisoning.

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Non-human toxicity values
Oral LD50 in rats: 2315 mg/kg
Oral LD50 in pigs: 550 mg/kg

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Toxicological information for (S)-Gossypol acetic acid is derived from studies on gossypol, which is known to exhibit dose-dependent toxicity. In animal studies, high doses of gossypol cause testicular toxicity, hepatotoxicity, and gastrointestinal effects. The compound can cause hypokalemia and has been associated with fertility effects in males. The (S)-enantiomer is considered less toxic than the racemic mixture at equivalent doses. For research use, standard laboratory precautions should be observed, including the use of personal protective equipment and working in a fume hood. The compound should be stored at -20degC, protected from light and moisture. Waste containing gossypol derivatives should be disposed of according to institutional guidelines.

[1]. In vitro effects of the BH3 mimetic, (-)-Gossypol, on head and neck squamous cell carcinoma cells. Clin Cancer Res. 2004 Nov 15;10(22):7757-63.

Therapeutic Uses
/Experimental Therapy/ Gossypol (C(30)H(30)O(8)) is a polyphenolic compound derived from the cotton plant (Malvaceae family, Gossypium genus). The gossypol molecule contains six phenolic hydroxyl groups and two aldehyde groups, giving it high chemical activity. Gossypol can undergo Schiff base formation, ozone decomposition, oxidation, and methylation reactions to generate various gossypol derivatives. Due to the diverse biological activities of gossypol and its derivatives, including antifertility, antiviral, anticancer, antioxidant, antitrypanosomiasis, antibacterial, and antimalarial activities, it has been a focus of numerous studies. Because the rotation of the naphthalene ring interphase is restricted, gossypol is a chiral compound with two transisomers (i.e., (+)-gossypol and (-)-gossypol), which exhibit different biological activities. Gossypol is a small-molecule Bcl-2 family pro-survival protein inhibitor and has been shown to inhibit the growth of AI prostate cancer. However, in a mouse model of prostate cancer xenograft (vertebral prostate cancer [VCaP]) treated with AT-101 (R-(-)-gossypol acetate), the presence of androgens attenuated the apoptotic effects of gossypol. This study aimed to better understand the in vitro effects of the androgen receptor (AR) on AT-101-induced apoptosis. VCaP cells treated with AT-101 exhibited increased apoptosis and downregulated expression of the pro-survival protein Bcl-2. Combined treatment with AR activation and AT-101 reduced apoptosis, increased cell survival, and attenuated caspase activation. AT-101 downregulated the expression of Akt and the apoptosis inhibitor X (XIAP), while AR stimulation restored the expression of these proteins. Combined treatment with bicalutamide and AT-101 increased apoptosis by reducing the expression of these pro-survival proteins. These data suggest that combined treatment with AT-101 and ADT may further delay the onset of AI disease, thereby prolonging progression-free survival in prostate cancer patients.
/Experimental Treatment/...A series of new and known gossypol bis-Schiff base analogs were synthesized, and their anticancer activity against HeLa, U87, and M85 cells was tested. Results showed that less active (+)-gossypol could be converted into more active derivatives through simple chemical modification. Many more potent compounds were found compared to (-)-gossypol, which may be promising anticancer drugs; some of these compounds showed superior activity against all three cancer cell lines compared to the anticancer drug cisplatin.../Gossypol Analogs/
/Experimental Treatment/ Twenty-seven patients with pathologically confirmed gliomas relapsed after radiotherapy were treated with gossypol 10 mg orally twice daily. Of these, 15 had glioblastoma, 11 had anaplastic astrocytoma, and 1 had recurrent low-grade glioma. Efficacy was assessed every 8 weeks using CT/MRI scans and clinical criteria, including dexamethasone requirements. Treatment continued until disease progression. Two patients achieved partial remission (PR); four patients remained stable for 8 weeks or longer. One patient maintained PR with improved KPS score for 78 weeks. Another patient achieved partial remission for 8 weeks. Toxicity was mild: two patients with prior extensive treatment experienced mild thrombocytopenia, five patients experienced hypokalemia, and three patients experienced grade 2 hepatotoxicity and peripheral edema. In this study, gossypol levels determined by high-performance liquid chromatography (HPLC) were not correlated with efficacy or toxicity. We conclude that gossypol is well tolerated and, although its efficacy is low in a population of patients with recurrent gliomas who have received extensive treatment and have a poor prognosis, it is measurable…
For more complete data on the therapeutic uses of gossypol (7 items in total), please visit the HSDB record page.

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Drug Warning
Following clinical trials conducted in China in the 1970s, gossypol was proposed for use as a male contraceptive. This review summarizes numerous formal animal toxicology studies on gossypol and the recovery of fertility in men after discontinuation of gossypol treatment. These studies prompted the World Health Organization (WHO) Special Programme for Research, Development and Research Training in Human Reproduction (HRP) to decide that gossypol is unsuitable as an anti-fertility drug. …Reports indicate that studies conducted in China have confirmed the effectiveness of gossypol as an anti-fertility drug for men. …Research by the International Organization for the Advancement of Chemical Sciences (IOCSD) showed that 40 out of 70 novel high-purity gossypol structural forms had no higher activity than pure gossypol. Experiments in Sprague-Dawley rats and cynomolgus monkeys confirmed that both (-) and (+) gossypol are too toxic for human contraception. Among the side effects associated with gossypol use, the most serious is hypokalemic paralysis, although the reported differences in incidence may be attributed to variations in dietary potassium intake across different regions and genetic susceptibility. On the other hand, two independent studies confirmed the findings regarding the risk of permanent infertility in healthy men of reproductive age, finding an irreversible infertility rate of 25%. Failure to recover after discontinuing gossypol may be related to prolonged treatment duration, high total dose of gossypol, small testicular volume, and elevated follicle-stimulating hormone (FSH) levels...

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(S)-Gossypol acetic acid (CAS#: 1189561-66-7) is a potent and selective BH3 mimetic that targets anti-apoptotic Bcl-2 family proteins. The molecular formula is C32H34O10 with a molecular weight of 578.61. The acetic acid salt (1:1 stoichiometry) is formed by treating gossypol with acetic acid. (S)-Gossypol has been extensively investigated in preclinical studies as a potential anticancer agent, particularly for Bcl-2-overexpressing hematological malignancies and solid tumors. The compound has entered clinical trials as an anticancer therapeutic. It is also used as a chemical probe to study apoptosis regulation and BH3-only protein biology. The compound is supplied for research use only and is not approved for therapeutic use.

C32H34O10

578.606370449066

578.215

1189561-66-7

(R)-(-)-Gossypol acetic acid;866541-93-7;Gossypol (acetic acid);12542-36-8;(R)-(-)-Gossypol;90141-22-3

3503

Light yellow to yellow solid powder

184 °C (from ether); 199 °C (from chloroform); 214 °C (from ligroin)
178 - 183 °C

6

8

5

38

780

0

NIOHNDKHQHVLKA-UHFFFAOYSA-N

InChI=1S/C30H30O8.C2H4O2/c1-11(2)19-15-7-13(5)21(27(35)23(15)17(9-31)25(33)29(19)37)22-14(6)8-16-20(12(3)4)30(38)26(34)18(10-32)24(16)28(22)36;1-2(3)4/h7-12,33-38H,1-6H3;1H3,(H,3,4)

acetic acid;7-(8-formyl-1,6,7-trihydroxy-3-methyl-5-propan-2-ylnaphthalen-2-yl)-2,3,8-trihydroxy-6-methyl-4-propan-2-ylnaphthalene-1-carbaldehyde

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~86.41 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.32 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)